Aberrant proteolysis, a hallmark of many pathologies, is often localized to certain organs and tissues. Unfortunately, basic studies to understand spatial aspects of aberrant proteolysis in human disease models are difficult to carry out, because current inhibitors lack the ability to be activated in a location- and time-specific fashio. Furthermore, even though small molecule inhibitors are available to target aberrant proteolysis, inhibition in non-target tissues can be a significant cause of side effects and toxicity during chemotherapeutic interventions. In general, we must be able to not only measure, but also inhibit proteolysis in a spatially controlled fashion in human disease models, in order to study spatial aspects of aberrant protease activity. Towards this goal we have developed an exciting new method that gives spatial and temporal control over enzyme inhibition using light. Our photoactivated approach works against isolated enzymes and in live cells, and targets an important class of cysteine proteases associated with inflammation and cancer progression. We developed an effective caging method based on neutralizing nitrile warheads of protease inhibitors through metal binding, where fast inhibitor release and activation occur with visible light, without causing deleterious toxicity. During this grant period, we propose to make a significant step towards our long-term goal, to develop and apply a complete toolkit of light-activated inhibitors to investigate the role of cysteine proteases in inflammation and cancer in live animal models. Specific Aims are 1) to develop new, potent versions of caged cathepsin and caspase inhibitors, 2) to understand the photochemistry of caged protease inhibitors and tune the wavelength of release; 3) to characterize the behavior of caged complexes in living cells; 4) to achieve local inhibition of cathepsin and caspase enzymes in a murine model of bone tumor growth. We have assembled a highly complementary and interdisciplinary research team for this project that has a proven track record of working and publishing together. Targeting of bone tumors was chosen, because bone is the primary site of metastasis for prostate cancer and most patients with advanced disease experience complications from bone lesions that are incurable. This proposal aims to develop and validate light-activated cysteine protease inhibitors as basic research tools, and potential therapeutics in metastatic bone disease. Literature data confirm that photodynamic therapy is effective against bone tumors in live animal models, which strongly supports our studies.